International Journal of Food Sciences and Nutrition, Volume 55, Number 4 (June 2004) 325 /331

Effect of temperature on soluble invertase activity, and glucose, fructose and sucrose status of onion bulbs (Allium cepa) in store
Noureddine Benkeblia, Shuichi Onodera, Taiki Yoshihira, Shinichi Kosaka and Norio Shiomi
Department of Food and Nutrition Sciences, Graduate School of Dairy Science Research, 582 Bunkyodai Midorimachi, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan

The activity of soluble invertase, and the variation in glucose, fructose and sucrose contents in onion bulbs (Allium cepa ) during long-term storage at 108C and 208C were investigated. Invertase activity increased progressively after 8 weeks to 0.084 and 0.092 nkat/g fresh weight (FW), then sharply to 0.29 and 0.35 nkat/g FW at 208C and 108C, respectively, and remained high during 5 weeks. Then, activity decreased abruptly to 0.039 and 0.041 nkat/g, and remained low during the last 8 weeks and close to that observed initially. Glucose increased to 17.73 and 14.62 mg/g FW after 4 weeks at 208C and 108C, respectively, then decreased sharply between week 5 and week 7 to 4.13 and 4.91 mg/g FW, respectively, and remained rather stable ranging from 9 and 10 mg/g FW at both temperatures. Fructose showed a similar pattern and was 14.8 and 21.68 mg/g FW at 208C and 108C, respectively. Between week 10 and week 24, fructose ranged from 5 and 6 mg/g FW, and from 6 and 7 mg/g FW at 208C and 108C, respectively. Sucrose increased to 19.63 and 14.43 mg/g FW at 208C and 108C, respectively, decreased during 3 weeks, and then increased randomly from 5.69 to 9.42 mg/g FW at 208C, but remained in a steady state at 108C ranging 5.039/0.78 mg/g FW. During the last 6 weeks, the sucrose content was higher at 208C than at 108C. The fructose/glucose ratio varied during the first 8 weeks but remained at a steady level during the last 16 weeks. The (glucose'/fructose)/sucrose ratio increased randomly at 108C, whereas at 208C the ratio increased during 10 weeks then decreased progressively during the final 14 weeks.

Introduction Bulb dry matter content is an important quality parameter of onion, and several investigations have attempted to relate bulb characteristics and storage life (Rutherford & Whittle, 1984). About 80% of bulb dry matter is non-structural carbohydrates (Darbyshire & Henry, 1981). The predominant of these non-structural carbohydrates are glucose, fructose, sucrose and low-molecularweight fructans, while starch and raffinose

Correspondence to: N. Benkeblia, E-mail: ben-nour@rakuno.ac.jp

ISSN 0963-7486 printed/ISSN 1465-3478 online # 2004 Taylor & Francis Ltd DOI: 10.1080/09637480412331290512

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are absent (Darbyshire & Henry, 1981; Benkeblia et al ., 2002). The metabolism of sugars is closely linked to the dormancy and sprouting states (Kato, 1966), and the most important biochemical changes occurring during long-term storage of bulbs, as with other vegetables, are related to the quantitative variation in the carbohydrate constituents. Variation in carbohydrate changes in onion bulbs during storage were reported by numerous authors (Rutherford & Whittle, 1982; Hurst et al ., 1985; Benkeblia et al ., 2002; Benkeblia & Varoquaux, 2003); however, the relationship between variation of sugars and physiological and environmental factors is not clearly understood despite being well established. Surprisingly, the status of saccharides in stored onion bulbs and its relationship with invertase activity and temperature has not been studied, despite the metabolic importance of sucrose, which is an intermediate compound between fructans and trisaccharides, on one hand, and glucose and fructose, on the other. Invertase (b-D-fructofuranosidase; EC 3.2.1.26) is a highly polymorphic protein that catalyses the irreversible cleavage of sucrose to glucose and fructose, which are then available for cellular activity such as growth development and maintenance, respiration and cell wall synthesis. The plant kingdom contains multiple forms of invertase that can be distinguished on the basis of optimum pH, glycosilation state, solubility and subcellular location (Tang et al ., 1996; Koch & Zeng, 2002). Although invertases were examined in a large number of plants (Sturm, 1999), their role in plants is complex and depends critically upon subcellular location (Tang et al ., 1996). In many sink tissues, invertase is closely correlated with growth (Arai et al ., 1991) and expansion (Schaffer, 1986) of the tissues. According to von Schaewen et al . (1990), invertase might contribute to the control of hexose formation in leaves, interrupts export of sucrose and leads to an accumulation of carbohydrates. On the other hand, little is known about the expression in stored organs, particularly edible bulbs. In this paper we report the activity of soluble invertase and the sugar

variation in long-term stored onion bulbs under two temperatures. Materials and methods Onion bulbs Onion bulbs (Allium cepa cv. Tenshin) freshly harvested and dried in the field for 2 weeks were obtained from the local farm of the university. They were sorted for uniformity and absence of defects, packed in commercial plastic (PVC) trays each of 15 kg. Storage conditions Onion bulbs were stored in the dark at two temperatures. The onions were placed in a refrigerated chamber (Eyelatron, model FLI 301 NH; Rikakikai, Tokyo, Japan) at 109/0.18C and 709/1% relative humidity. At 208C, onions were placed in a ventilated room with an average relative humidity of 55%. Three trays were stored at each temperature. Soluble invertase extraction Soluble invertase was extracted according to the method of van den Ende et al . (1998), and all operations were carried out on ice. Tissues (20 g) were homogenised in 40 ml ice-cold 50 mM Na-acetate buffer (pH 5) containing 10 mM NaHSO3, 1 mM PMSF, 5 mM 2-mercaptoethanol and 10 mM mannitol as internal standard, using a blender at full speed for two 30 sec intervals. The homogenate was squeezed through three layers of cheesecloth and centrifuged at 15,000 )/g for 15 min. The supernatant was then collected, and filled up to 40 ml with the extraction buffer. An aliquot of 10 ml was concentrated to 1 ml and purified by centrifugation (3000 )/g during 1 h, five times) using an Amicon CentriPrep centrifugal YM 10 filter (Amicon Bioseparation, Millipore, Bedford, MA, USA). This enzyme extract was used for the invertase assay. Invertase assay The enzymatic reaction was carried out in a total volume of 0.4 ml containing sodium acetate buffer (pH 5) containing 0.02% (w/v) Na-azide. Invertase activity was assayed by

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incubating an aliquot of enzymatic extract (0.1 ml) in 50 mM sodium acetate buffer (0.1 ml) and substrate solution containing 200 mM sucrose (0.2 ml). The reaction mixture was incubated at 308C for 2 h, and stopped by heating in a boiling bath for 5 min. The fructose content formed was determined by high-performance anion exchange chromatography (Dionex, Sunnyvale, CA, USA) as described later. One unit of invertase is defined as the amount of invertase that releases one micromole of fructose per minute under the assay conditions. The invertase assay was run in triplicate and the results were expressed as nkatal per gram of fresh weight (FW). Saccharide extraction Saccharides (glucose, fructose and sucrose) were extracted as described by Shiomi (1992). Tissues (10 g) were homogenised in 80 ml aqueous ethanol (70%) containing a small amount of calcium carbonate. The homogenate was then boiled under reflux in a water bath for 10 min. Then homogenate was filtered and the residue extracted three times with aqueous ethanol and one time with water in the same conditions. The filtrates were combined and filled up to 500 ml with distilled water. An aliquot of the filtrate (10 ml) was concentrated under vacuum at 358C to dryness using a Buchi rotavapor. The concentrated sugars were collected in 1 ml water and passed through a 0.45 mm filter and analysed by high-performance anion exchange chromatography (Dionex). All processes were run in triplicate. Saccharide analysis The saccharides were separated on a highperformance liquid chromatography (HPLC)-carbohydrate column PA1, Carbo Pack with a Dionex Bio LC series HPLC and a pulsed amperometric detector (PAD). The gradient was established by mixing eluent A (150 mM NaOH) with eluent B (500 mM Na-acetate in 150 mM NaOH) in two ways */system I: 0/1 min, 25 mM; 1/2 min, 25 /50 mM; 2 /20 min, 50/200 mM, 20/22 min, 500 mM; 22 /30, 25 mM; and system II: 0/1 min, 5 mM; 1/2 min, 25/50

mM; 2/14 min, 50 /500 mM, 14 /22 min, 500 mM; 22 /30, 25 mM. The flow rate through the column was 1.0 ml/min. The applied PAD potentials for E1 (500 ms), E2 (100 ms) and E3 (50 ms) were 0.01, 0.06 and /0.6 V, respectively, and the output range was 1 mC. Statistical analysis All determinations were carried out in triplicate and the experiment was repeated twice. Results were expressed on a fresh weight basis, and data were averaged and compared by Student t-test using Statistica 5.0 software.

Results and discussion Invertase activity in stored onion bulbs is shown in Figure 1. Immediately after storing at 108C the invertase activity increased slightly in onion tissues, whereas at 208C the activity increased after 3 weeks. After 7 weeks the activity progressively reached 0.089 and 0.092 nkat/g FW at 208C and 108C, respectively. Then, invertase activity increased sharply to 0.29 and 0.35 nkat/g FW and remained high during 5 and 6 weeks at 208C and 108C, respectively. This activity decreased abruptly to 0.039 and 0.041 nkat/g FW, and then increased slightly a second time to 0.11 and 0.12 nkat/g FW at 208C and 108C, respectively, after 16 weeks. During the final 8 weeks, the invertase activity decreased

Figure 1. Effect of temperature and storage time on invertase activity in onion bulbs: k, 208C; m, 108C.

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slowly to 0.033 and 0.058 nkat/g FW at 208C and 108C, respectively. The variation of invertase activity in onion bulbs has not been reported previously; however, the different peaking observed during storage suggests that invertase tends to cause an accumulation of hexoses, which will be continuously supplied to the meristematic tissues favoring sprout development, and providing substrates for growth at different sucrose-importing sites (sinks). Similar results were observed in lily bulbs, where invertase activity was high after 2 weeks at 48C and 108C (Shin et al ., 2002). Zrenner et al . (1996) reported also that invertases are involved in the regulation of the hexose-tosucrose ratio in cold-stored potatoes. The glucose content increased from 9.31 to 17.73 and 14.62 mg/g FW after 4 weeks at 208C and 108C, respectively, then decreased sharply between weeks 5 and 7 to 4.13 and 4.91 mg/g FW, respectively (Figure 2a). A slight increase to 9.79 and 8.43 mg/g FW at 208C and 108C, respectively, was observed during week 7 and week 11. After, and during 13 weeks, glucose content remained rather stable, ranging from 9 and 10 mg/g FW at both temperatures. The fructose content showed a similar pattern to glucose, and after 4 weeks the content was 14.8 and 21.68 mg/g FW at 208C and 108C, respectively (Figure 2b). After 4 weeks, the fructose content variation was similar to glucose variation, and between week 10 and week 24 the content ranged from 5 and 6 mg/g FW, and from 6 and 7 mg/g FW at 208C and 108C, respectively. The fructose/glucose ratio decreased during the first 2 weeks, then increased after 4 weeks and was 1.01 and 1.22 at 208C and 108C, respectively (Figure 3). After a decrease was noted between week 5 and week 8, the fructose/glucose ratio remained at a steady level during the final 16 weeks, averaging 0.689/0.06 and 0.839/0.07 at 208C and 108C, respectively. This high level of the fructose/glucose ratio observed at refrigerated temperature is due to the low cellular metabolism slowed down by low temperature. This relative weak metabolism leads to a slight accumulation of fructose even if glu-

Figure 2. Glucose (a) and fructose (b) content variation in stored onion bulbs: k, 208C; m, 108C.

Figure 3. The fructose/glucose ratio in stored onion bulbs: k, 208C; m, 108C.

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cose consumption is also lower at a refrigerated temperature. Glucose and fructose variations in onion bulbs during storage have been the subject of many investigations. However, large intervariabilities and intravariabilities were observed depending on cultivar and storage conditions. In most previous investigations, a peak of glucose and fructose was reported but at different time scales. The peaks were observed between 1 and 3 months (Hurst et al ., 1985; Bottcher, 1992; Croci et al ., 1995; Benkeblia et al., 2002). The present results suggest that glucose and fructose variations seem to be less dependent on temperature, and are consistent with the claim of Benkeblia et al . (2002), but in contrast with finding of Hurst et al . (1985) who reported that temperatures had an effect on the sugar changes. Nevertheless, the variation in glucose and fructose levels in onion is not clearly elucidated, and depends on numerous factors, particularly cultivar, sugar content and the dormancy release time, which affect largely the metabolism of sugars during the break of dormancy and the onset of spouting. The sucrose content variation is shown in Figure 4. During the first 4 weeks, sucrose increased progressively and was 19.63 and 14.43 mg/g FW at 208C and 108C, respectively. Then, the sucrose content decreased 3 three weeks to 4.72 and 3.31 mg/g FW. respectively. Between weeks 8 and 18, sucrose

increased randomly from 5.69 to 9.42 mg/g FW at 208C, whereas its content at 108C remained in a steady state, ranging 5.039/0.78 mg/g FW. During the final 6 weeks, sucrose increased to an averaged value of 13.579/0.76 at 208C, whereas at 108C it increased slightly to 8.64 mg/g FW and decreased again. During the final 10 weeks of storage, we noted that sucrose content was higher at 208C than at 108C. This could be the consequence of high fructans degradation and a high relative catabolism level, which needs substantial substrate quantity. The (glucose'/fructose)/sucrose ratio show ed two distinguished states, as shown in Figure 5. During the first weeks of storage, the (glucose'/fructose)/sucrose ratio decreased from 2.29 to 1.31 and 2.08 at 208C and 108C, respectively. Then, this ratio increased to 2.93 after 6 weeks at 208C, and to 2.73 after 4 weeks at 108C. At 108C, the ratio increased to 3.29 and remained stable between week 11 and week 18, then decreased sharply to 1.91. Three weeks later, it increased a second time to 3.15 and remained high. On the other hand, at 208C this ratio decreased during the last 14 weeks to a final value of 1.21. These variations suggest that at 108C glucose and fructose contents are high due to the low catabolism causing their slight accumulation, and this suggestion is emphasised by the fructose/glucose ratio observed during the same period. Comparatively, at 208C glucose and fructose contents are low

Figure 4. The sucrose content variation in stored onion bulbs: k, 208C; m, 108C.

Figure 5. The (fructose'glucose)/sucrose ratio in stored / onion bulbs: k, 208C; m, 108C.

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due to the high substrate consumption that is a consequence of the sprout development of the bulbs. The variation in glucose, fructose and sucrose levels in onion bulbs has not been extensively investigated. According to Salama et al . (1990), the glucose content of onions increased slightly during the first 5 weeks at 08C and 158C, and then gradually decreased at 0, 15 and 308C. On the other hand, the fructose content increase during storage at 0 and 158C, while at 308C it decreased after 20 weeks. The sucrose content decreased slightly at 0 and 158C but increased slightly at 308C. Similar results on fructose were reported by Rutherford & Whittle (1982) at 08C; however, Hurst et al . (1985) noted a decrease in total sugars of onion kept during 6 months at 1 and 48C, but no variation was noted at 218C. Benkeblia et al . (2002) also did not observe any significant difference in total saccharides (glucose, fructose and sucrose) of onion bulbs stored for 6 months at 4, 10 and 208C. The variations in glucose, fructose and sucrose levels reported by these numerous investigations suggest that catabolism of sucrose is not completely elucidated (Winter & Huber, 2000; Koch & Zeng, 2002). This component plays a central role in growth and development in plants,

and demand for products of sucrose degradation are important factors particularly during the break of dormancy when internal sprouting could be initiated by the accumulated disaccharides (Benkeblia & SelseletAttou, 1999). Finally, it is concluded from the results of this investigation that invertase activity and sucrose catabolism were less influenced by temperature and seem to be more under the influence of the physiological state of the bulb. The first peaks of invertase and sugars observed could be an indicator of the dormancy release and regrowth of sprouts, which may lead to a greater demand for mobilisation and utilisation of glucose and its metabolic origin sucrose involving invertase. However, further investigations are needed to determine, on one hand, the flux and sugar status within the different scales of the bulbs, and on the other hand the activity of other degrading enzymes of carbohydrates in onion bulbs.

Acknowledgements */The nancial support of this work is provided by JSPS Japan, under a Postdoctoral Fellowship. Partial support by a Grant-in-Aid for Promotion of High Technology Center Project Research from the Ministry of Education, Government of Japan is also acknowledged.

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